A broad spectrum of eukaryotic viruses, including adenoviruses, adeno-associated viruses, Herpes viruses and retroviruses, has been used to express genes in cells. Each type of vector has demonstrated a viral-dependent combination of advantages and disadvantages. Accordingly, careful consideration must be given to the advantages and disadvantages inherent to a particular type of vector when deciding which vector should be used to express a gene.
Adenoviruses are advantageous because they are easy to use, can be produced in high titers (i.e., up to about 10.sup.13 viral particles/ml), transfer genes efficiently to nonreplicating, as well as replicating, cells (see, for example, review by Crystal,Science 270: 404-410 (1995)), and exhibit a broad range of host- and cell-type specificity. Such advantages have resulted in a recombinant adenovirus being the vector of choice for a variety of gene transfer applications. Adenoviral vectors are especially preferred for somatic gene therapy of the lungs, given their normal tropism for the respiratory epithelium.
Other advantages that accompany the use of adenoviruses as vectors in vivo gene expression include: (1) the rare observance of recombination; (2) the absence of an ostensible correlation of any human malignancy with adenoviral infection, despite the common occurrence of infection; (3) the adenoviral genome (which is comprised of linear, double-stranded DNA) can be manipulated to carry up to about 7.5 kb of exogenous DNA, and longer DNA sequences can potentially be carried into a cell, for instance, by attachment to the adenoviral capsid (Curiel et al., Human Gene Therapy 3: 147-154 (1992)); (4) an adenovirus can be modified such that it does not interfere with normal cellular function, given that the vector controls expression of its encoded sequences in an epichromosomal manner; and (5) it already has been proven safe to use in humans, given that live adenovirus has been safely used as a human vaccine for many years.
Using adenoviral reporter gene constructs, it has been established that high levels of gene expression can be obtained in a variety of animal models. However, it also has been established that the high level of gene expression so obtained is transient, with reporter gene expression peaking within the first week after infection and becoming essentially undetectable about 80 days after infection. Recent studies have indicated that the limited persistence of gene expression in vivo is most likely due to an immune response of the host against violably infected cells. For example, gene expression can be maintained in immunologically privileged neuronal or retinal tissues for periods in excess of two months and in immunodeficient or immunologically naive rodents for periods in excess of six months.
Intravenous administration of adenovirus to mice results in the vast majority of adenovirus being localized to the liver (Worgall et al., Human Gene Therapy 8: 37-44(1997)). During the first 24-48 hrs of infection, 90% of vector DNA is eliminated, presumably through innate pathways of viral clearance mediated by Kupffer cells in the liver (Worgall et al. (1997), supra), well before maximal levels of transgene are expressed. In spite of the fact that the majority of virus is cleared within one to two days, over 95% of hepatocytes are transduced by the remaining small percentage of input adenoviral vectors (Li et al., Human Gene Therapy 4: 403-409 (1993)) with maximum transgene expression occurring during the first week of post-infection. Transgene expression, however, rapidly declines to baseline levels in immune-competent animals within 2-3, weeks of infection due to immune activation.
Using a combination of mouse strains, which are defective in specific elements of the immune system, it has been shown that the immune response against cells infected with viral vectors involves both cellular and humoral components of the immune system. For example, immunodeficient mice, which lack mature T- and B-lymphocytes express adenovirus-mediated transgenes beyond four months (Kass-Eisler et al., Gene Therapy 1: 395-402 (1994); Yang et al., Immunity 1: 433-442(1994a); Yang et al., PNAS USA 91: 4407-4411 (1994b); Dai et al., PNAS USA 92: 1401-1405 (1995); Kay et al., Nat. Genet. 11: 191-197 (1995); and Yang et al., J. Immunol. 155: 2564-2570 (1995)). Similarly, transfer of CD8.sup.+ and CD4.sup.+ cytotoxic T-cells from adenoviral vector-infected mice to infected RAG-2 mice, which lack mature B- and T-cell lymphocytes, results in clearance of the vector and the transgene by apoptosis (Yang et al. (1994a), supra; and Yang et al. (1995), supra), whereas immune depletion of CD8.sup.+ or CD4.sup.+ cells in immunocompetent mice results in persistent transgene expression (Yang et al. (1994a), supra; Kay et al., Nat. Genet. 11: 191-197 (1995); Yang et al. (1995), supra; Kolls et al., Hum. Gene Ther. 7: 489-497(1996); and Guerette et al., Transplantation 62: 962-967 (1996)). While pathways involving perforin and Fas are the major pathways responsible for T-cell cytotoxicity (Kojima et al., Immunity 1: 357-364 (1994); Henkart, Immunity 1: 343-346 (1994); Kagi et al., Science 265: 528-530 (1994); and Kagi et al., Eur. J. Immunol. 25: 3256-3262 (1995)), the perforin/granzyme pathway has been reported to mediate clearance of adenoviral gene transfer vectors by antigen-specific, cytotoxic T-cells (Yang et al., PNAS USA 92: 7257-7261 (1995)).
In addition to limiting the persistence of gene expression from viral vectors, the immune response inhibits successful readministration of viral vectors, which limits the period of gene expression. For example, adenoviruses are classified into 47 different serotypes and a number of subgroups, namely A through G, based on a number of criteria, including antigenic cross-reactivity. Following an initial administration of adenovirus, serotype-specific antibodies are generated against epitopes of the major viral capsid proteins, namely the penton, hexon and fiber. Given that such capsid proteins are the means by which the adenovirus attaches itself to a cell and subsequently infects the cell, such antibodies are then able to block or "neutralize" reinfection of a cell by the same serotype of adenovirus. This necessitates using a different serotype of adenovirus in order to administer one or more subsequent doses of exogenous DNA to continue to express a given gene, such as in the context of gene therapy.
Various methods of inhibiting an immune response to vectors, such as viral vectors, in particular adenoviral vectors, have been proposed. One such approach involves the introduction of substantial deletions in a viral vector so as to reduce or eliminate completely the production of viral antigens by the viral vector. In this regard, the deletion of E4 from adenoviral vectors is especially important for safe vector design. Removal of the E4 region severely disrupts viral gene expression in transduced cells. Removal of the E4 region also eliminates several viral products that interact with and antagonize cellular targets and processes. E4-ORF6 has been shown to block p53 function and to have oncogenic potential (Dobner et al., Science 272: 1470-1473 (1996); Nevels et al., PNAS USA 94: 1206-1211 (1997)). It also appears that E4-ORF1 has oncogenic potential (Javier et al., J. Virol. 65: 3192-3202 (1991); Javier et al., Science 257: 1267-1271 (1992); Javier et al., Breast Cancer Res. Treat. 39: 57-67 (1996); Javier et al., J. Virol. 68: 3917-3924 (1994); Weiss et al., J. Virol. 71: 4385-4393 (1997); Weiss et al., J. Virol. 71: 1857-1870 (1997); and Weiss et al., J. Virol. 70: 862-872 (1996)). ORF6 and ORF3 of the E4 region of adenovirus also have been shown to be involved in altering mRNA expression post-transcriptionally (Nordqvist et al., PNAS USA 87: 9543-9547 (1990); Nordqvist et al., Mol. Cell. Biol. 14: 437-445 (1994); Nordqvist et al., Mol. Biol. Rep. 14: 203-204 (1990); Ohman et al., Virology 194: 50-58 (1993); Sandler et al., J. Virol. 63: 624-630 (1989); and Sandler et al., Virology 181: 319-326 (1991)). E4 products are also involved in controlling E2F (Nevins, Virus Res. 20: 1-10 (1991)), E1A-induced p53-independent apoptosis (Marcellus et al., J. Virol. 70: 6207-6215 (1996)), the modulation of the phosphorylation status of cellular and viral proteins (Kleinberger et al. 67: 7556-7560 (1993); and Muller et al., J. Virol. 66: 5867-5878 (1992)), and the alteration of the nuclear transport of various proteins (Goodrum et al., J. Virol. 70: 6323-6335 (1996)). Elimination of the E4 region of adenovirus eliminates these negative effects. However, E4 elimination also adversely affects maintenance of transgene persistence.
Provision of E4 in trans has been proposed as a method of activating transgene expression from an E4.DELTA. adenoviral vector (Brough et al., J. Virol. 71(12): 9206-9213 (1997)). Supply of E4 products in trans has been demonstrated to allow persistent expression from the cytomegalovirus E4 promoter (Armentano et al., J. Virol. 71(3): 2408-2416 (1997)). Co-expression of the adenoviral E2 preterminal protein from an adenoviral vector or in trans has been demonstrated to stabilize in vitro an adenoviral mini-genome, which is deficient in E1, E2 and E3 but not E4 (Lieber et al., Nature Biotech. 15: 1383-1387 (1997)). Expression of a transgene operably linked to the cytomegalovirus immediate early promoter has been demonstrated to be dependent on the infected cell protein 0 in Herpes simplex vectors; based on such a showing, it was suggested that ORF3 of the E4 region of adenovirus could have the same effect on transgene expression in an adenoviral vector (Samaniego et al., J. Virol. 72(4): 3307-3320 (1998)).
The present invention seeks to address some of the disadvantages inherent to the methods and vectors of the prior art by providing, among other things, methods and vectors that modulate the persistence of expression of a transgene in an at least E4deficient (E4.DELTA.) adenoviral vector. This and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the following detailed description.